Proliferation stimulants and process of making same



are found to stimulate Patented Aprr 22, 1941 uraasr ron am AND PRO PROCESS OF MAKING George Sperti. muons-emu rue DIM mu M Foundation, CincinlI-tLOJalmamatlcncfOhlo ,No Drawing; Application June 19, 1 939, Serlal No. 279,997

Claims.

This invention relates to the extraction from cellular matter of either animal or plant origin of an active fraction or extract having metabolic stimulating properties for cellular matter and more particularly the capacity to stimulate cellular proliferation, although other properties may Gil also be present such as the capacity to stimulate cellular respiration and to promote reactions such as fermentation. This application is a continuation in part of my prior application Serial No. 184,337, filed January 10, 1938.

It is known that when cellular matter such as yeast or. malt combingsis. destroyed by suspension in a toxic medium such as alcohol or by heat, a product is obtained which among other properties has the capacity to stimulate growth or proliferation of living yeast. The composition or constitution of such products, however, and their mode of action, have not been fully understood. In my cop'ending application, Serial No. 280,000, filed June 19, 1939, concurrently herewith, I have disclosed the production from cellular matter of extracts having the capacity to stimulatecellular respiration, these extracts being prepared from the cellular matter by various processes. While these extracts are primarily useful for the stimulation of respiration, some of them' nevertheless also cellular proliferation or growth.

The present invention involves the discovery that if thecells are treated insuch a manner as to injure the cells or interfere with their normal life processes and such as to extend such harmful of for substantial periods of time as distinguished (Cl. lea-w tained with yeast, rat, mouse and chick embryo,

rat spleen and liver, various types of bacteria, etc. For conveniencein treating the cellular matter, and also to facilitate the subsequent separation of the active principle from the remaining cellular matter, which is usually desirable but not always necessary, it is preferred with some start-.

ing materials, such as yeast, malt combings, and

- the like, to form a suspension of the cellular matter in a suitable non-toxic or substantially nontoxic liquid such as water, isotonic salt solutions such as Ringer's solution, nutrient media, alcohol in high dilution (of the order of 10% or less), tc. In this form the cells are treated so as to cause injury or harm to their normal life processes but the treatment is regulated so as to prevent rapid ultra-violet radiation, x-rays, etc., the intensityof the treatment being regulated to prevent instant or substantially instant destruction of the cells. Chemical injuring agents, highfrequency agitation or other mild but sustained mechanical injury can also be used. The amount of active material increases as the time of treatment is prolonged. On the other hand, if the treatment is too mild, it may not affect the cells throughout the whole mass undertreatment and may therefore require an impracticable length of treatment from rapid or immediate destruction, the product produced is both quantitatively andqualitatively different from growth factors produced as disintegration products. The possibility is thereby suggested that the active principle responsible for proliferation-promotingproperties is in some way produced or given oil as a physiologic response to injury to the life processes of the cells,

as distinguished from disintegration products that would bereleased on destruction of the cells, but whatever may be the true explanation, proliferation-promoting properties which differ both quantitatively and qualitatively have been found to result from the process-disclosed and claimed hereinafter. V 1

Living cellular matter of widely varying nature and origin and from both the animal and plant kingdoms can be treated by a process embodying the invention to produce products as described in order to obtain practical yields. The intensity of the harmful treatment must therefore be correlated with the time required to obtain the desired amount' f active substance., It is' not necessary to destroy the cells but when conditions are such that the cells areslowly destroyed, an indication of the extent of completion of the process may be obtained by actual count ofthe dead cells as indicated by staining with methylene blue or by other standard method, .because when substantially all of the cells have been de- 'stroyed, further treatment has no further eifect.

After treatment the material may be eentm Y as by distillation, and the concentration may be 7 residue is obtained. above. For example, good results have been ob- The following examples are illustrative of the continued if desired until a substantially solid process described in generaiterms above, but are not to be construed as defining the limits of the invention, reference being had to the appended claims for this purpose.

Exsmrrn l Suspensions of Fleischma'nnsbakersyeast were I placed in quartz tubes which were then subjected to -ultra-violet irradiation from a quartz mercury arc, the distance of the tubes from the lamp being progressively increased so as to diminish the intensity of the treatment and the irradiation period being correspondingly prolonged until the same amount oi' killing of the yeast cells (90%) was obtained in each tube. The concentration of each suspension was 2 grams of yeast per 100 cc. of a 0.9% solution of NaCl.

lular matter was separated by filtration. filtrates were tested for growth-promoting properties by adding equal amounts thereof to cultures of yeast growing in Reader-s medium in rocker tubes ,for twenty-four hours-at 30 C. 4fter the growthperiod the yeast crop was determined by a; photo-densitometer. The efiect oi the filtrates was compared with the effects of similar filtrates from non-irradiated controls.

. Time of Distance m mp In irradiation, from lamp i Irradiated Non-irradiated 20 10 1.1 4e 40 17 1.3 .52 so 24 1. as .4 160 34 2. 1s .78

' Examrpn 2 tion. After irradiation. both suspensions were centrifuged or filtered through Biichner filters to remove the yeast, after which they were filtered through Berkefeld filters to free them from any terial so obtained, and subtracting the weight of the salt present. a

The materials were assayed by addingthem to cultures or yeast grown in Reader's medium in rocker tubes for twenty-four hours at 30 0. Each tube contained a'total or 25 cc. (including added material) .and was seeded with 0.064 mg. percc. of cultured yeast.

The-yeast crops after twenty-four hours were determined by a photoelectric photo-densitometer. Since this measured totalprotoplasm present rather than number of cells, the data are given in terms of yield on a weight basis (wet weight 01 yeast in mg. per cc.), rather than in population counts. Microscopic examination of the rocker-tube contentsafter the growth period showed, however, that the average size of cells was no greater than that in'the original inocula (frequently it was less because of the presence of many .young cells); hence increased crop weight represented stimulation of proliferation rather than oi! growth in cell size.

The proliferation-promoting potencies of the After f the irradiation was completed, the remaining cel- The : control suspemnsions.

. ulted an increase in remaining cells. The yields were. calculated by ms my taking the filtrates to dryness, w hing the masamples assayed are expressed in growth units, this unit being defined as an increased yeast crop alter twenty-four hours, corresponding to a count of 25 greater than that in the control tubes (count x 250=cells per cu. mm.). 0n the weight basis, this corresponded. to a concentration of 1.6 mg. per cc. greater than-that in the controls.v The number of growth units per mg. of material added to each cc. of rocker-tube. contents was calculated from curves relating the Conoenp Concen- Growth Prep: g Irradiatration Growth units ig arensions tion time of filunits per cc. um tion pa in in hours trate in per mg. of filg gg? v mg'lm mgJcc. trate Non-irradiated less than 1%stained Non- 0.59 1.39 0.82

0 stained in each case the oontrolsuspensioiis stood during the time of irradiation under s1m1la r conditions of teniperatme, etc. The difierenoes in time oi irradiatmn required for 90-100 per cent staining were obtained by v the depth and concentration of the suspe F irradiated. suspensions were filtered through Berkefeld filters The per cent of cells lcilled (stained) at the a end oi the irradiation period was the same in both instances. While in preparation B a suspension of only twicethe concentration'was used,

40 the yield per cc. of filtrate was approximately four times as great (with twice the amount of starting material) and the potency per mg. of

- material three times as great. Thus the ,yield in terms of growth units per-gram of yeast in v suspension was roughly six times as great in the long irradiation at low rate of injury. That this cannot be accounted for by the longer period vDiflerent methods of harmful treatment may I be combined ifdesired, the selection of the combination depending on conditions in each case. The-following example illustrates the combination of prolonged irradiation followed by more severe treatment with heat, and also shows the 4 better results obtained in thisway as compared with the more rapid injury resulting from a severe heat treatment followed by prolonged irradiation. Y

,Yeast suspensions were used containing 10 rams of bakers yeast per "100 cc. isotonic salt solution. One suspension was irradiated four hours and fifteen minutes by a Burdick-quartz mercury arc lamp operating at .volts. the suspension being placed in a quartz tube at adis- 70 tance of l5 cm. from the lamp. After this irra diation the suspension was boiled for three min- A'second suspension was treated in the e manner except that the boiling preceded 'the irradiation. In both cases the combined treatment resulted in completekilling of all cells.

nsions and from the second suspension being 7.91 mg, a per cc. The dry active material was then dis solved in distilled water at twice the concentration of the original filtrates. These solutions were tested for stimulation of growth of yeast by the procedure described above. The solutions were added to the rocker tubes in'increasing amounts expressed below in terms of sixteenths of 1 cc.:

Amount oi filtrate Irradiated,

Boiled. then then boiled irradiated I- i gees-z Exsu'ru: 4

Yeast suspensions comprising g. of bakers yeast per 100 cc. isotonic salt solution were placed in tubes which were mounted in an electromagnetic vibrator driven by means of a beat frequency oscillator at a frequency of 60 cycles per second. The treatment was continued for approximately ten hours, after which the suspensions were flltered, taken'to dryness, and then made up in solution in distilled water at twice the concentration of the original filtrates. These solutions were then tested for their capacity to stimulate the growth of yeast in rocker tubes according to the procedure described above. and

' compared at the same time with controls which Yeast crop alter 21 hours sas's'se in milligrams of test material per cc. of rocker tube contents. The results were as follows:

Yeast crop alter 24 hours in mg. per cc.

Irradiated Non-irradi- Irradiated Nomirradiyeast and yeast E. coli ated E. coli EXAMPLE 6 Suspensions of yeast were made in isotonic salt solution at concentrations of either 1.75 or 5 g. per 100 cc. These suspensions were divided into two portions, to one of which suflicient heteroauxin was added to give a concentration of 1 mg. of heteroauxin per cc. Both suspensions were allowed to stand for 24 hours, after which heteroauxin was added to the control suspension in concentration the same as that in the test suspension, and both were centrifuged and the supernatant fluids decanted. These fluids were dried and the dried materials were were not agitated. The results, expressed in growth units as defined in Example 2 above were Similar results have been obtained with other redissolved in distilled water at a concentration of 30 mg. per cc. (not including the weight of than by their relative proliferation-promotingefiects on an equal weight basis. These efiects were determined by adding varying amounts ofthe solutions to yeast cultures in rocker; tubes according to the procedure described above. the

amounts added being expressed below in mg. of the dried materials per cc. of the material in I the rocker tubes.

frequencies, particularly higher frequencies. and I with other types of agitation.

Exmu 5 I A yeast extract was made by the procedure described'above,"using 10 mg. of yeast per 100 cc.oi-isotonicsa.ltsohition,irradiatingwithultraviolet light, filtering and concentrating the fll trate. The yields from this yeast suspension and from a non-irradiated control yeast mm were respectively 2.88 mg. per cc. and 2.71 mg. percc.- 'lheseextractswerecomparedwithcorresponding extracts made from E. coli, grown in nutrient broth, irradiated in one case but not in the control. filtered and dried. These products were tested on yeast by adding varying amounts to yeast cultures in rocker tubes as described above, the amounts added being expressed below Yeast crop after 24 hours in my. per cc. I

H added to susn belorc Amount added Exnrrns'l' I Rat embryos were minced in a grinder and dividedintotwcportions. oneportionwassuspendedin Reader's solution and irradiated with a sperti quartz mercury arc'lamp at adlstance of 25 cm. for approximateifw minutes. The

tilled water to twice the concentration of the original nitrates. These solutionswere then tested by adding them to yeast cultures growing inrockertubesasdescribed-abovatheamoimts addedbeingbelcwinmmoifliedried Thomas tryptophane reagent.

ing activity was shown with Benedict's solution,

material per cc. of the material in the rocker tubes;

Yeast crop after 24 hours in mg. per cc.

Irradiated Non-insulated A -W Reader's Reader's By similar methods proliferation-promoting Substances can be obtained from yarious ammal tissues, such as chick and mouse embryo, rat spleen, rat'liver, beef spleen, etc.

In the foregoing examples, proliferation-promoting properties have been tested on yeast cultures, because this is a convenient and easily practiced method and because itis'a method in which the results can be readily determined and expressed in mathematical units. It has also been demonstrated in tissue cultures that these proliferation-promoting substances are capable of stimulating cellular proliferation of animal tissue. In such tests the amount of growth is indicated by the size of the cultured tissue after a' growth period. Marked stimulation of pro-. liferation has been observed when fractions from .yeast obtained as described above are added to embryo heart, chicken skin, and rat skin growing in tissue cultures. The extent of stimulation'in such cases, however, cannot be expressed mathematically. Growth factors from animal tissue such as'described in Example 7 have also been tested on tissue cultures with resulting stimulation of proliferation. Also growth factors from yeast and beef spleen have been tested clinically on human skin by incorporation in salves and have shown the same property of stimulating proliferation of epithelial cells.

It will be understood that these products may have other valauble properties than the promotion of proliferation. Oneis the capacity to stimulate respiration or oxygen absorption. When a small quantity of the yeast extract is added to yeast, for example, it is found that the" respiration of the yeast cells is stimulated and the oxygen absorption is increased. Stimulation of respiration of humanand animal skin can also be obtained through the use of such products,

, desoxypentoses (Kiliani test). The Thomas 13- naphtholtest'gave a blue ringwith both materials and a non-uniform brownish color with the Negligible reducand substantial freedom from proteinby the biuret test. Both materials gave positive murexide. Kossel, and diazo, and negative Weidel and 75' Wheeler-Johnston tests. The positive reactions .were more marked with the filtrates from irradiated suspensions. Slight turbidity or opalescence was obtained from phosphotungstic, plcric, and metaphosphoric acids. No precipitate formed with HgClz. No positive color reactions could be obtained for pyridine even after boiling. The active materials gave pH values of about 5.5 (glass electrode) in concentrations of 0.5 to 1.5 mg. per cc. Their activity was not reduced by autoclaving at 20' lb. for 15 minutes.

Biological tests also show the difference between these factors. and nucleic acids since both yeast nucleic and thymus nucleic acid are biologically inactive.

Thesechemical tests show thatthe growthpromoting factors are not nucleic acids, but

ultra-violet absorption spectra show these factors to be related or similar to nucleic acids.

The spectrum of the factor from injured cells (obtained by photographing the spectrum of the filtrate from irradiated yeast with filtrate from non-irradiated yeast'in the comparison cell and also the spectrum of the filtrate from irradiated rat liver) is characterized by a marked maximum at 2600 A. and a minimum at 2360 A similar to the spectra of nucleic acids and their derivatives. On irradiation of the filtrate from injured cells, the extinction at 2600 A. decreases and thatat 2360 A. and on the long-wave legof the curve increases, as ,in adenine, guanine, etc. Of ab-' sorbing components of nucleic acids, the spectrum least resembles that of uracil or cytosine, but bears a resemblance to guanine inthe broadness of the curveand to adenine in the 2600 AI maximum.

These factors are shown by biological tests to be distinguished from the following substances .present in yeast, all of which are biologically inactive: Vitamins B1 and B2 (thiamin and riboflavin), inositol, cystine, l-tryptophane, dl-xalanine, arginine and tyrosine. The spectrum of;

. these factorsis also markedly different from that of vitamin B1 and lacks the long-wave maximum o vitamin 132., These factors are also distinguished from nicotinic acid which fails to show stimulation growth of yeast. With growth-factors obtained by prolonged in- Jury in accordance with the above process, biological activity is correlated with absorption at 2690 A. No such correlation is obtained, how-' ever, .with the so-called bios.

Products such as described above which will stimulate and increase the rate of proliferation of growing yeast may be used to promote fermentation and similar reactions. Further, certam of these products have been found to stimulate and promote proliferation of human and animal skin and hence are valuable healing agents in topical remedies for cuts, burns, and various skin disorders.

It is to be understood that the foregoing examples areillustrative only and not limitative,

- as other 'types'of starting materials and other forms of harmful treatment may be employed. It is preferred for practical reasons to subject the cellular matter to an external treatment to cause the injury to the cells, but it will be understood that if the cells. are allowed to stand for 7. 'long periods under conditionswhich are not ideal," as for example in a-mildly toxic medium'or atunfavorable temperatures, some injury will be caused and some growth factor will be produced. The references above and in the appended claims to injury areto beunderstood to includetreatment where the cellular matter is held under conditions suiiiciently unfavorable as to produce useful quantities of the growth factor within practicable time limits.

What is claimed is:

l. The process of producing a substance capable of stimulating cellular metabolism which comprises subjecting living cells to treatment capable of destroying cellular life when applied in lethal dosage but limiting the intensity of said treatment to prevent immediate destruction of cells and continuing said treatment to cause the production of said substance by the living cells in response to said treatment, whereby the yield of said substance increases up to the point where substantially all the cells are destroyed, and

extracting the water-soluble constituents of the treated cells.

2. The process of producing a substance capable of stimulating cellular metabolism which comprises subjecting living cells in suspension in a substantially non-toxic medium to treatment capable of destroying cellular life when applied in lethal dosage but limiting the intensity of the treatment to prevent immediate destruction of cells and continuing said treatment to cause the production of said substance by the living cell in response to said treatment, whereby the yield of said substance increases up to the point where substantially all the cells are destroyed, and rea moving the solid matter from the suspension to obtain a cell-free liquid containing said substance.

3. The process of producing a substance capable of stimulating cellular metabolism which comprises subjecting-living cells in suspension in a substantially non-toxic medium to treatment capable oi! destroying cellular life when applied in lethal dosage but limiting the intensity 'of the treatment to prevent immediate destruction of cells and continuing said treatment tocause the production of said substance by the living cell in response to said treatment, wherebythe yield of said substance increases up to theP int where vsubstantially all the cells are destroyed, and

removing the solid matter irom'the suspension to obtain a cell-tree liquidcontalning said substance and concentrating said liquid to recover said substance in solid tom.

4. The process of producing a substance capable of stimulating cellular metabolism which 5. The process of producing a substance capable of stimulating cellular metabolism which comprises subjecting living cells in suspension in a substantially non-toxic medium to irradiation with ultraviolet light while limiting the intensity of the radiation to prevent immediate destruction of cells and continuing the irradiation to cause the production of said substance by the living cells in response to the irrad ation, whereby the yield of said substance increases up to the point where substantially all the cells'are destroyed, and removing the solid matter from the suspension to obtain a cell-free liquid. containing said substance.v

6. The process of producing a substance capable of stimulating cellular metabolism which comprises subjecting living yeast cells to treatment capable of destroying cellular life when applied in lethal dosage but limiting the intensity of said treatment to prevent immediate destruction of cells and continuing said treatment to cause the production of said substance by the living cells in response to said treatment, whereby the yield of said substance increases up to the point where substantially all the cells are destroyed,

and extracting the water-soluble constituentsoi the treated cells. 7 I

7. The process of producing a substance capable of stimulating cellular metabolism which comprises subjecting living yeast cells in suspension in a substantially non-toxic medium to irradiation with ultraviolet light while limiting the intensity of the radiation to prevent immediate destruction of cells and continuing the irradiation to cause the production oi! said substance by the living cells in response to the irradiation, whereby the yield of said substance increases up to the point where substantially all the cells are destroyed, and removing the solid matter from the suspension to obtain a cell-free liquid containing said substance.

8. The process of producing a substance c'apa-.

. ble of stimulating cellular metabolism which comcomprises irradiating living cells with ultraviolet light while limiting the intensity or the radiation to prevent immediate destruction or cells and continuing the irradiation to cause the production or said substance by the living cells in response to the irradiation, whereby the yield oi. said substance increases up to the point where substantially all the cells are destroyed, and extracting the water-soluble constituents oi the irradiated 60 duced cells.

prises subjecting living liver cells in suspension in a substantially non-toxic medium to treatment capable of destroying cellular life when applied in lethal dosage but limiting the intensity of the treatment to prevent-immediate destruction of cells and continuing said treatment to cause the production of said substance by the living cell in response to said treatment, whereby the yield of said substance increases up to the point where substantially allthe cells are destroyed, and removing the solid matter from the suspension to obtain a cell-tree liquid containing said substance.

9. A stimulant for cellular metabolismproduced by the process defined in claim 1.

10; A stimulant for cellular metabolism proby the process defined in claim 2.

GEORGE SPER'I'I. 

